In the recently published paper “Anion dependence in the partitioning between proton and electron transfer in ion/ion reactions” by J. J. Coon et al., Int. J. Mass Spectrom. 236, 33-42, (2004), the reactions of multiply charged positive ions (cations) with specific classes of negative ions (anions) in linear ion traps are analyzed. Linear ion traps (also termed 2D ion traps, because the electric fields in the interior only change in two dimensions) comprise four rods, to which an RF voltage (radio frequency voltage) is applied, with end electrodes which repel the ions. The authors describe which types of anion lead only to a simple deprotonation (“charge stripping”) of organic biopolymers, and which types of anions primarily result in electron transfer, which leads to subsequent cleavages of the backbone of these biopolymers with high yield (ETD=electron transfer dissociation). The fragment ions here belong to the so-called C and Z series, and are therefore very different to the fragment ions of the B and Y series, which are obtained by collisionally induced fragmentation (CID). The fragments of the C and Z series have advantages for the identification and determination of the amino acid sequence from the mass spectrometric data.
The authors' linear ion trap was specially equipped for the simultaneous storage of positive and negative ions. It had grids at both ends, which were operated with RF voltages and could therefore repel ions of both polarities. In addition, the positive ions were introduced from one end, the negative ions from the other end, and could initially be kept apart in the linear ion trap by special measures which generated an axial DC potential profile before the reaction was started by switching off the DC potential profile. The setup of the linear ion trap was therefore much more complex than that of commercial instruments.
The authors report in the cited paper that acquainted well-known scientists had not been able to detect any electron transfer, or the associated fragmentation, in 3D ion traps, even in reactions with the same combinations of cations and anions, which would have led to electron transfer in linear ion traps. The positive ions were introduced into the 3D ion trap in the usual way through an aperture in one of the two end caps, and the negative ions through an aperture in the ring electrode. The authors speculate in a separate section 3.7 of the cited article (titled “3D versus 2D traps for ETD”) about the reasons why electron transfer cannot occur in 3D ion traps. One of the explanations is that the ions in a 3D ion trap are confined from all sides by pseudopotential fields, whereas in 2D ion traps they would have freedom of movement in one direction. So electron transfer dissociation (ETD) in 3D ion traps was not only not detected, despite searching, but authors who are very experienced in this field, and must be taken seriously, are also discussing the fact that electron transfer cannot occur in 3D ion traps and why this is so.
Three-dimensional ion traps (3D ion traps) according to Wolfgang Paul comprise a ring electrode and two end cap electrodes. The ring electrode is usually supplied with a one-phase RF voltage while the end cap electrodes are basically grounded but other modes of operation are also possible. In the interior of the ion trap, ions can be stored in the quadrupole RF field. In principle, 3D ion traps are better suited for reactions between positive and negative ions, because positive and negative ions can be stored simultaneously without any redesign of the trap, in contrast to commercial linear 2D ion traps with repelling end electrodes which repel only either positive or negative ions and cannot store both types of ions simultaneously without some complicated redesign.
The ion traps can be used as mass spectrometers by mass-selectively ejecting the stored ions mass by mass and measuring them with secondary-electron multipliers. Several different methods of ion ejection have been described, but they will not be discussed further here. More exact the word “mass” should be read by “mass-to-charge-ratio m/z” throughout this description, because only the mass m divided by the number z of elementary charges is effective in all kinds of mass spectrometry. Sometimes the mass-to-charge-ratio m/z is called “specific mass”, meaning the charge specific mass or charge related mass.
The maximum RF voltage at the ring electrode is very high, between 15 and 30 kilovolts (peak-to-peak) for customary ion trap mass spectrometers. The frequency is around one megahertz. In the interior of the 3D ion trap, essentially an RF quadrupole field is generated, which oscillates with the RF voltage and drives the ions above a threshold mass to the center, causing these ions to execute so-called secular oscillations in this field. The restoring forces in the ion trap are usually described by a so-called pseudopotential, which is determined by a temporal averaging of the forces of the real potential on the oscillating ions. The pseudopotential increases uniformly and quadratically in all directions and is effective for both polarities of ions. The ions oscillate in this “well” of the pseudopotential.
The ions can be generated in the interior or be introduced from outside. A collision gas in the ion trap ensures that the oscillations of the ions which are present at the onset are decelerated in the well of the pseudopotential; the ions then collect as a small cloud in the center of the ion trap. The diameter of the cloud in normal ion traps with normal ion fillings of a few thousand ions is around one millimeter; it is determined by an equilibrium between the restoring force of the pseudopotential and the repelling Coulomb forces between the ions. The internal dimensions of commercially available 3D ion traps are usually characterized by distance between the end caps of around 14 millimeters; the diameter of the ring is around 14 to 20 millimeters.
Ion trap mass spectrometers have properties enabling them for many types of analysis. In particular, selected ion species (so-called “parent ions”) can be isolated and fragmented in the 2D or 3D ion trap. Isolation of an ion species means that all ion species which are not of interest are removed from the ion trap by strong resonant excitations or other measures, so that only the parent ions remain. The fragmentation is brought about by a weak resonant excitation of the ion oscillations with a dipolar alternating voltage across the two end cap electrodes of the 3D ion trap (or across two electrode rods in case of 2D ion traps), which leads to many collisions with the collision gas, without removing the ions from the ion trap. The ions can collect energy in the collisions, which finally leads to the decomposition of the ions. For the fragmentation, one normally starts with doubly charged parent ions. In the prior art of ion traps, the ions have only been fragmented by such collisions with collision gas (CID=collision induced dissociation). The spectra of these fragment ions are called “daughter ion spectra” or “fragment ion spectra” of the selected parent ions concerned. Structures of the fragmented ions can be read off from these daughter ion spectra; it is therefore possible (although difficult) to determine the sequence of the amino acids of a peptide from these spectra. “Granddaughter ion spectra” can also be measured as fragment ion spectra of selected, isolated and fragmented daughter ions.
A widely used method of ionizing large biomolecules is to use electrospray ionization (ESI), which ionizes ions at atmospheric pressure outside the mass spectrometer. These ions are then introduced into the vacuum of the mass spectrometer, and from there into the ion trap, by means of inlet systems of a known type. RF ion guides are usually used to transfer the ions within the vacuum system, these ion guides usually taking the form of hexapole or octopole rod systems.
This ionization by electrospray ionization generates hardly any fragment ions. The ions are mostly those of the protonated molecule. But it is the strength of electrospray ionization that lots of multiply charged ions of the molecules are formed (doubly and triply charged ions). The lack of almost any fragmentation during the ionization process limits the information from the mass spectrum to the molecular weight; there is no information concerning internal molecular structures that can be used for further identification of the substance present. This information can only be obtained by acquiring fragment ion spectra.
A new method for fragmenting biomolecules, predominantly peptides and proteins, was described some years ago in ion cyclotron resonance or Fourier transform mass spectrometry. It consists in capturing low kinetic energy electrons from usually doubly charged ions. The electron capture mechanism leads to breaks of the backbone of the usually chain-shaped molecules. The method is called ECD (electron capture dissociation). If the molecules were doubly charged, one of the two fragments created remains as an ion. The fragmentation follows very simple rules (for specialists: there are essentially only an exceptionally large number of C cleavages, a small number of Z cleavages and very few Y cleavages between the amino acids of a peptide), so that it is relatively simple to elucidate the structure of the molecule from the fragmentation pattern. It is often very simple to read off the sequence of peptides or proteins directly from the mass differences of the exceptionally large C fragment ion signals; in contrast to the evaluation of collisional fragmentation spectra. It is significantly easier to interpret these ECD fragment spectra than CID fragment spectra. In addition, ECD fragment ions do not lose side chains like those formed by post translational modifications, whereas CID spectra do regularly lose these side chains. Thus the ECD spectra contain complementary information to that of CID spectra; it is particularly useful to have both types of fragment spectra available for evaluation.
It is also possible to fragment triply charged ions in this way, but the method is particularly impressive when used with doubly charged ions. If electrospray ionization is applied to peptides, the doubly charged ions are generally also the most prevalent ions. Electrospray ionization is a method of ionization that is very frequently used for biomolecules for the purpose of the mass spectrometric analysis in ion traps.
Fragmentation by electron transfer in reactions between multiply charged cations and suitable anions, as discussed above, would be a suitable alternative to electron capture dissociation (ECD), which is very difficult to carry out in ion traps, since the RF fields scarcely permit the entry of low-energy electrons. Fragmentation by electron transfer produces fragments which are very similar to those produced by electron capture.